The base games currently has main 18 biomes and 5 boss biomes while the DLCs add another 6 main biomes and 4 boss biomes, with plans to add more. The order in which you'll encounter biomes, and the general location of their links, are predetermined but they are divided into levels and only one biome of each level can be accessed in a run which gives players a lot of flexibility and change each run.
Within each run, biomes are procedurally generated within the confines of that biome's set parameters. This puts the focus of gameplay on a combination of on the moment adaptation and memorization, whith each biome having a distinct feel.
Each biomes also has a set of enemies that the player will encounter, some of them are unique to a specific biome. Higher difficulties change the enemy distribution up.
Scaling and difficulty
Each tier and biome has their own scaling of enemy and trap stats with tiers which operate somewhat differently compared to scaling of player damage and health with Stats. The exact formula used for scalig is not known at the moment but the tiers used for these are meaning we can determine which biomes are easier and which are harder.
Shops and Treasures
Excluding boss biomes, each biome always contains combinations of shops and treasures. The majority of these are predetermined but their type might be random, shops might be either be a weapons or skills shop and treasure rooms could contain treasure chests, linked altars or very rarely an additional cursed chest. Their locations are always random except for the fact they will always have the same access requirements.
Playing on higher difficulty using collected Boss Stem Cells grants you access to boss cell doors which can contain more shops, treasure rooms, cursed chests or even new exits.
Access and Exits
Each biome has exits leading to the biomes of the next level. Some of these can accessed without any requirement but most are blocked off by rune requirement, which need to be found and unlocked first. The locations of exits in abiome is mostly random, with some exceptions, but are always at the end of hallways or level chunks. They are also seperated, meaning that if there are multiple exits a biome splits up creating different paths depending on the biome.
Additional higher difficulties also unlock new exits allowing new paths.
The collector's room
After exiting each biome, the player will enter the Collector's room. Here, they can turn in blueprints, spend cells, enter Timed and Perfect Doors if they met the requirements, re-roll and upgrade gear, choose and change mutations and replenish their health and health potions. Collector's rooms are different depending on which biome you exited and entered with some of them having secrets.
After exiting a boss biome the player will enter a version of the Collector's room which also holds The Blacksmith and a perfect kill door.
The player can open a biome map that will show everything the player has explored in the biome. Shown on the map are activated teleporters, shops and treasure rooms.
The player can switch to a world map view. Shown here are discovered biomes in color with their names and undiscovered biomes in gray without their name. A biome counts as discovered when it has been visited while an undiscovered biome is shown from the moment you enter a biome that can exit into that biome. The position of the player is marked with a flaming head. The location of the player is not shown when inside the collector's room. Hightlighting a discovered biome will show previously travelled paths between it and biomes of the previous and next tier. The path used in the current run is shown in a gray line. The world map will also show the next biome that has the incentivized biome bonus.
Castle Key Doors: They are 6 doors of 3 different colors, 2 doors each. Each 2 doors lead to an extra section of the biome, which offers an Elite enemy fight and a Castle Key as reward.
Castle Key: 3 in total. 2 are required to access Throne Room, while the 3rd gives access to blueprint of Boomerang and a Scroll of Power. Dropped by the three Elite enemies in each of the Castle Key doors.
Technically inaccessible stage, entrance was behind the door that required 5 boss cells to open in the Graveyard. If accessed via modifying the boss cell counter with third-party tools, it could be seen that it had the same 'darkness' mechanic as the Forgotten Sepulcher. Only enemies present in the stage appeared to be Cannibals. The enemy tier appears to be absurdly high. The majority of the stage was locked behind a door that required a key (simply named "Key") to open, which can't be spawned even by third party modifiers
An old ending point of a run that persisted until the 1.0 update came around. It would be accessed after defeating the final boss of the game, which at first was Conjunctivius, then the Time Keeper, and finally, the Hand of the King. It used to have the Fisherman NPC right next to a boat, who would impale the Beheaded with a tentacle to let the player start a new run. Later on, that was changed to a "Work in Progress" sign and a tube, through which the Beheaded would crawl to go back to the Prisoners' Quarters.